Measurements of Flavin‐Containing Monooxygenase (FMO) Activities

Randy L. Rose1

1 North Carolina State University, Raleigh, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.9
DOI:  10.1002/0471140856.tx0409s13
Online Posting Date:  November, 2002
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Abstract

Measurement of Flavin‐Containing Monooxygenase (FMO) Activities (Randy L. Rose, North Carolina State University, Raleigh, North Carolina). This unit describes methods used for measuring the presence of flavin‐containing monooxygenases using NADPH oxygenation and methamizole oxidation. Methods are also provided to determine the relative contributions of FMO versus cytochrome P450 from microsomes.

     
 
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Table of Contents

  • Basic Protocol 1: NADPH Oxidation
  • Basic Protocol 2: Methimazole/DTNB Assay
  • Basic Protocol 3: Determinations of FMO Versus CYP Contributions to Metabolism for Common Substrates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: NADPH Oxidation

  Materials
  • 2× tricine/KOH buffer, pH 8.5 (see recipe)
  • 10 mM nicotinamide adenine dinucleotide phosphate, reduced form (NADPH; see recipe)
  • Enzyme (usually purified or recombinant FMO)
  • 0.1 M substrate (see recipe for suggestions on preparation and storage)
  • 1.5‐ml UV grade (methacrylate) cuvettes, 1‐cm path‐length
  • Spectrophotometer
  • 37°C water bath

Basic Protocol 2: Methimazole/DTNB Assay

  Materials
  • 2× tricine/KOH buffer, pH 8.5 (see recipe)
  • 1 mM 5,5′‐dithio‐bis(2‐nitrobenzoic acid) (DTNB; see recipe)
  • 10 mM nicotinamide adenine dinucleotide phosphate, reduced form (NADPH; see recipe)
  • 2 mM dithiothreitol (DTT; see recipe)
  • 100 mM methimazole (see recipe)
  • Inhibitors (optional)
  • Microsomes
  • Enzyme
  • Spectrophotometer
  • 37°C water bath
  • 1.5‐ml UV grade (methacrylate) cuvettes, 1‐cm path‐length

Basic Protocol 3: Determinations of FMO Versus CYP Contributions to Metabolism for Common Substrates

  Materials
  • Microsomes
  • 100 mM potassium phosphate buffer, pH 7.4 (see recipe)
  • NADPH regenerating system (see recipe)
  • Substrate (see recipe for suggestions of preparation and storage)
  • Antibodies to NADPH cytochrome P450 reductase (BD Biosciences)
  • 2× tricine/KOH buffer, pH 8.5 (see recipe)
  • 1 mM N‐benzylimidazole (NBI; prepare from 300 mM stock; see recipe)
  • 50°C water bath
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Figures

Videos

Literature Cited

Literature Cited
   Atta‐Asafo‐Adjei, E., Lawton, M.P. and Philpot, R.M. 1993. Cloning, sequencing, distribution, and expression in Eschericia coli of flavin‐containing monooxygenase 1C1. J. Biol. Chem. 268:9681‐9689.
  Blake, B.L., Rose, R.L., Mailman, R.B., Levi, P.E., and Hodgson, E. 1995. Metabolism of thioridazine by microsomal monooxygenases: relative roles of P450 and flavin‐containing monooxygenase. Xenobiotica 25:377‐393.
   Burnett, V.L., Lawton, M.P., and Philpot, R.M. 1994. Cloning and sequencing of flavin‐containing monooxygenases FMO3 and FMO4 from rabbit and characterization of FMO3. J. Biol. Chem. 269:14314‐14322.
  Cashman, J.R., Bi, Y.‐A., Lin, J., Youil, R., Knight, M., Forrest, S., and Treacy, E. 1997. Human flavin‐containing monooxygenase form 3: cDNA expression of the enzymes containing amino acid substitutions observed in individuals with trimethylaminuria. Chemic. Res. Toxicol. 10:837‐841.
  Cherrington, N.J., Cao, Y., Cherrington, J.W., Rose, R.L., and Hodgson, E. 1998. Physiological factors affecting protein expression of flavin‐containing monooxygenases 1, 3 and 5. Xenobiotica 7:673‐682.
  Devereux, T.R., Philpot, R.M., and Fouts, J.R. 1977. The effects of Hg2+ on rabbit hepatic and pulmonary solubilized, partially purified N,N‐dimethylaniline N‐oxidases. Chem. Biol. Interact. 15:277‐287.
  Dixit, A. and Roche, T.S. 1984. Spectrophotometric assay of the flavin‐containing monooxygenase and changes in its activity in female mouse liver with nutritional and diurnal conditions. Arch. Biochem. Biophys. 233:50‐63.
  Elfarra, A.A. 1995. Potential role of the flavin‐containing monooxygenases in the metabolism of endogenous compounds. Chem.‐Biol. Interact. 96:47‐55.
   Falls, J.G., Blake, B.L., Cao, Y., Levi, P.E., and Hodgson, E. 1995. Gender differences in hepatic expression of flavin‐containing monooxygenase isoforms (FMO1, FMO3, and FMO5) in mice. J. Biochem. Toxicol. 10:171‐177.
  Falls, J.G., Ryu, D.‐Y., Cao, Y., Levi, P.E., and Hodgson, E. 1997. Regulation of mouse liver flavin‐containing monooxygenases 1 and 3 by sex steroids. Arch. Biochem. Biophys. 342:212‐223.
  Grothusen, A., Hardt, J., Brautigam, L., Lang, D., and Bocker, R. 1996. A convenient method to discriminate between cytochrome P450 enzymes and flavin‐containing monooxygenases in human liver microsomes. Arch. Toxicol. 71:64‐71.
   Hodgson, E., Blake, B.L., Levi, P.E., Mailman, R.B., Lawton, M.P., Philpot, R.M., and Genter, M.B. 1995. Flavin‐containing monooxygenases: Substrate specificity and complex metabolic pathways. In Molecular Aspects of Oxidative Drug Metabolizing Enzymes: Their Significance in Environmental Toxicology, Chemical Carcinogenesis and Health (E. Arinc, J.B. Schenkman, E. Hodgson, eds.) pp. 225‐235, NATO ASI Series, Vol. H90, Springer‐Verlag, Berlin Heidelberg.
   Hodgson, E., Cherrington, N., Coleman, S.C., Liu, S., Falls, J.G., Cao, Y., Goldstein, J.E., and Rose, R.L. 1997. Flavin‐containing monooxygenase and cytochrome P450 mediated metabolism of pesticides: From mouse to human. Rev. Toxicol. 2:231‐243.
  Kinsler, S., Levi, P.E., and Hodgson, E. 1988. Hepatic and extra‐hepatic microsomal oxidation of phorate by the cytochrome P450 and FAD‐containing monooxygenase systems in the mouse. Pestic. Biochem. Physiol. 31:54‐60.
   Kinsler, S., Levi, P.E., and Hodgson, E. 1990. Relative contributions of the cytochrome P450 and flavin‐containing monooxygenases to the microsomal oxidation of phorate following treatment of mice with phenobarbital, hydrocortisone, acetone and piperonyl butoxide. Pestic. Biochem. Physiol. 37:174‐181.
  Lawton, M.P., Gasser, R., Tynes, R.E., Hodgson, E., and Philpot, R.M. 1990. The flavin‐containing monooxygenase enzymes expressed in rabbit liver and lung are products of related but distinctly different genes. J. Biol. Chem. 265:5855‐5861.
  Lawton, M.P., Cashman, J.R., Cresteil, T., Dolphin, C.T., Elfarra, A.A., Hines, R.N., Hodgson, E., Kimura, T., Ozols, J., Phillips, I.R., Philpot, R.M., Poulsen, L.L., Rettie, A.E., Shephard, E.A., Williams, D.E., and Ziegler, D.M. 1994. A nomenclature for the mammalian flavin‐containing monooxygenase gene family based on amino acid sequence identities. Arch. Biochem. Biophys. 308:254‐257.
  Sabourin, P.J., Myser, B.P., and Hodgson, E. 1984. Purification of the flavin‐containing monooxygenase from mouse and pig liver microsomes. Int. J. Biochem. 16:713‐720.
  Schlenk, D. 1998. Occurrence of flavin‐containing monooxygenases in non‐mammalian eukaryotic organisms. Comp. Biochem. Physiol. C. Pharmacol. Toxicol. Endocrin. 121:185‐195.
   Tynes, R.E. and Hodgson, E. 1985. Catalytic and substrate specificity of the flavin‐containing monooxygenase in microsomal systems: Characterization of the hepatic, pulmonary and renal enzymes of the mouse, rabbit, and rat. Arch. Biochem. Biophys. 240:77‐93.
  Tynes, R.E., Sabourin, P.J., and Hodgson, E. 1985. Identification of distinct hepatic and pulmonary forms of microsomal flavin‐containing monooxygenase in mouse and rabbit. Biochem. Biophys. Res. Commun. 126:1069‐1075.
  Venkatesh, K., Levi, P.E., and Hodgson, E. 1991. The effect of detergents on the purified flavin‐containing monooxygenase of mouse liver, kidney and lungs. Gen. Pharmacol. 22:549‐522.
  Williams, D.E., Hale, S.E., Muerhoff, A.S., and Masters, B.S.S. 1985. Rabbit lung flavin‐containing monooxygenase. Purification, characterization and induction during pregnancy. Molec. Pharmacol. 28:381‐390.
  Williams, D.E., Ziegler, D.M., Nordin, D.J., Hale, S.E., and Masters, B.S.S. 1984. Rabbit lung flavin‐containing monooxygenase is immunologically and catalytically distinct from the liver enzyme. Biochem Biophys. Res. Commun. 125:116‐122.
   Zeigler, D.M. 1980. Flavin‐containing monooxygenases: Catalytic mechanism and substrate specificities. In Enzymatic Basis of Detoxication (W.B. Jakoby, ed.), vol.1, pp. 201‐225, Academic Press, New York.
  Zeigler, D.M. 1988. Flavin‐containing monooxygenases—Catalytic mechanism and substrate specificities. Drug Metabolism Reviews 19:1‐32.
  Zeigler, D.M. and Mitchell, C.H. 1972. Microsomal oxidase 4. Properties of a mixed‐function amine oxidase isolated from pig liver‐microsomes. Arch. Biochem. Biophys. 150:116‐125.
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